Effects Of VEGF On Expression Of TFPI In Bone Marrow Mesenchymal Stem Cells And Aorta Of The Model Of Atherosclerosis

BackgroundAtherosclerosis (AS), a kind of general arterial disease, is a leading dangerous disease over the world with high morbidity and mortality. The clinical manifestation of AS includes myocardial infarction, stroke, aortic aneurysm,aortic dissection as well as intermittent lameness. The etiopathogenisis of this disease, however, need to be further determined, which leads to unsatisfactory curative effect.Therefore, the study of its etiopathogenisis and effective treatment is very significant.Vascular Endothelial Growth Factor (VEGF) has diverse biologic activities, including promoting proliferation of endothelial cells, angiogenesis, synthesis of vasoactive substances, vasodilation, anti-thrombokinesi and inhibition of smooth muscle cells (SMCs) proliferation and so on. Notedly, promoting proliferation of endothelial cells by VEGF presents high speciality.Interestedly, VEGF performs as a double-edged sword during the development of AS. On the one hand, VEGF displays anti-AS through such mechanisms as endothelial protection by increasing nitrogen monoxidum and prostacyclin-2, inhibiting SMCs proliferation, anti-thrombosis, and especially remedial angiogenesis, which provids a novel pathway for anti-AS. On the other hand, VEGF maybe aggravate vascular injury via accelerating atherosclerotic plaques. Therefore, it is very significant to further illustrate the precise mechanism underlying atheroprotection.Thrombogenesis is a leading cause among all the diseases of the mortality of Chinese, which usually develops in atherosclerotic vessels and induces necrosis of organ and tissue involved. Although the therapy for anti-thrombosis has made increasing progress, it still need further study for its effect and safety. Tissue factor pathway inhibitor (TFPI) is regarded as a very essential natural anticoagulant substances in vivo, which has been demonstrated that TFPI can inhibit thrombogenesis and may be a promising anti-thrombotic agent.Bone marrow mesenchymal stem cells (BMMSCs) is a sort of adult stem cells with high proliferative and differentiated potency, which can differentiate into various mesenchymal cells such as osteoblasts, chondroblasts,lipoblast and alike. Recently, BMMSCs was observed to differentiate into cadiocytes,neurocytes and vascular endothelial cells and so on. BMMSCs not only have common merits as other stem cells such as self renewal and multi-directional differentiating ability, but also hold personal chatraterifies including less-injury, non-age-limitation, non-immunologic-rejection, easily-getting, easy-culture-in-vitro and so on, all of which make BMMSCs as a ideal engineering cell in the field of cell therapy and gene therapy.RNA interference (RNAi) technique has intrigued extensive interest since it was discovered for its high performance, low toxin and high specificity. As a novel and effective tool for gene function analysis, RNA interference (RNAi) technique has been developed rapidly and made great progress in diverse study fields including plant, eumycete, worm, inferior vertebrate studies and even mammalian cells, for its rapid and accurate gene-kickout. A lot of studies made great breakthrough via inhibiting expression of certain genes by means of this technique. It has been demonsrated to reduce cost of RNA synthesis by means of vector-mediated intra-cellular mRNA delivery, which will promote RNAi widely used.VEGF is viewed as an important factor for the maintenance of edothelial function and TFPI plays a crucial role for anti-coagulation. It has been well documented that VEGF is markedly relative to TFPI. The mechanism underlying the interaction between VEGF and TFPI, however, has not been studied. Therefore, we will investigate their relationship and mechanism involved for a novel effective anti-AS therapy, i.e. BMMSCs vector-mediated gene therapy.ObjectiveThis series of experiments was to construct and identify a recombinant eukaryotic expression plasmid carrying human vascular endothelial growth factor gene and short interfering RNA eukaryotic expression vector for VEGF.MethodsHuman VEGF cDNA was amplified with polymerase chain reaction and was inserted into the eukaryotic expression vector pcDNA3.1(-). The recombinant plasmid pcDNA3.1(-)VEGF was identified with PCR, double enzyme digestion and DNA sequencing. A VEGF-siRNA targeting human VEGF mRNA common sequence was synthesized and was inserted into BamHI,Hindâ…¢linearized pSilencer^TM neoU₆2.1 vector. The sequence of pU-VEGF-siRNA recombinant plasmid was analyzed by DNA sequencer.ResultsThe recombinant plasmid pcDNA3.1(-)VEGF was confirmed with PCR, double enzyme digestion and DNA sequencing. It was verified that the sequence of constructed recombinant plasmid pU-VEGF-siRNA was correct by DNA sequencing. We obtained cells with stable expressions of VEGF and VEGF-siRNA.ConclusionThe recombinant plasmid pcDNA3.1(-)VEGF carrying human vascular endothelial growth factor was successfully constructed. It indicated that hairpin siRNA eukaryotic expression vector for VEGF would be successfully established.ObjectiveIn this study we firstly established the method of in vitro culture and expansion of infant human bone marrow mesenchymal stem cells and investigated their biological characteristics and multipotential capacity. Then, we identifed the human bone marrow mesenchymal stem cells (BMMSCs) and investigated the expression of VEGF mRNA and TFPI mRNA in BMMSCs. Finally, we investigated the changes of the level of VEGF and TFPI protein in BMMSCs transfected with pcDNA3.1 (-)VEGF and pU-VEGF-siRNA recombinant eukaryotic expression plasmid.Methods1. BMMSCs were isolated and purified from infant human bone marrow by percoll density gradient centrifugation, and were cultured and amplified in vitro by cell culture with low glucose DMEM containing 15% fetal bovine serum. The biological characteristics and multipotential capacity of the BMMSCs were observed. 2. The pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid were transiently transfected into BMMSCs being cultured in vitro with Lipofectamine 2000. RT-PCR methods were used to detect the expression of VEGF mRNA and TFPI mRNA, Westem blot methods were used to detect the levels of VEGF and TFPI protein and MTT to detect the biological activity of VEGF of the conditioned medium after the transfection. 3. The pU-VEGF-siRNA recombinant eukaryotic expression plasmid were transiently transfected into BMMSCs being cultured in vitro with Lipofectamine 2000. RT-PCR methods were used to detect the expression level of VEGF mRNA and TFPI mRNA and Western blot methods were used to detect the levels of VEGF and TFPI protein.Results1. The biological characterizations of human BMMSCs in long term culture: The bone marrow mononuclear cells were firstly cultured for 72 hours in primary culture. Cells achieved confluence when the BMMSCs incubated for one weeks. At sub-confluence the cells were passaged, split,and replated. The BMMSCs were extremely purified after this cycle had been repeated five times. The cells had a fibroblastic-like morphology and were well spread out. The cells continued to maintain this morphology with further passaging and were homogeneous in appearance;80%-90% of the BMMSCs became confluent between 10 and 12 days in primary culture; The growth curves of passage 1, 5, 9and 15 were quite similar (p＞0.05); The adhesive rates of passage 1, 5, 9and 15 were very much similar (p＞0.05); The effects of different serum concentration and cell number plated on the proliferation of BMMSCs were tested by MTT method. The proliferation index of BMMSCs was increased in 2.5%-20% concentration of serum in a dose dependent manner, and was not further increased when the concentration of serum reached 20%. The most appropriate concentration of serum was 20%. The proliferation of BMMSCs was increased in the density of 3-10×10⁴/ml in a dose dependent manner. The most appropriate density of cell number was 10×10⁴/ml; The BMMSCs were resuscitable after frozen,and more than 85% of the BMMSCs were alive. 2. The BMMSCs could differentiate into osteoblasts, chondrocytes, adipocytes on special conditions in vitro,which accorded with the golden criterion for the BMMSCs. 3.Significantly high transient transfection efficiency in the BMMSCs can be achieved with the help of cation lipofectin mediation by fluorescent microscopy. There were no significant differences in the expression of VEGF mRNA and TFPI mRNA in the BMMSCs transfected with and without pcDNA3.1(-) vector (Control group). But the expression of VEGF mRNA and TFPI mRNA was increased in the BMMSCs transfected with pcDNA3.1(-)VEGF plasmid groups (p＜0.05 vs Control group), while transfection with pU-VEGF-siRNA plasmid significantly decreased the expression of VEGF and TFPI mRNA in the BMMSCs (p＜0.05 vs Control group). There were no significant differences in the biological activity of VEGF in cells without transfection, transfected with pcDNA3.1(-) vector, and transfected with pU-VEGF-siRNA plasmid groups(p＞0.05), However, The biological activity of VEGF in cells transfected with pcDNA3.1(-)VEGF plasmid groups was significantly increased (p＜0.05 vs Control group). 4. There were no significant differences in the level of VEGF protein and TFPI protein in cells without transfection and transfected with pcDNA3.1(-) vector (p＞0.05). But the level of VEGF and TFPI protein in cells transfected with pcDNA3.1(-)VEGF plasmid was significantly upregulated (p＜0.05 vs Control group), while the level of VEGF and TFPI protein of the BMMSCs transfected with pU-VEGF-siRNA plasmid groups was significantly down-regulated (p＜0.05 vs Control group). Conclusion1. The BMMSCs is purified cells, which can maintain its undifferentiating and the stable biological characterizations for the long term. The growth curves and adhesive rates of passage 1, 5, 9 and 15 are quite similar. The BMMSCs is multipotentia, which can differentiate into osteoblasts, chondrocytes, adipocytes on special conditions in vitro. 2. The expression of VEGF mRNA, TFPI mRNA, VEGF protein and TFPI protein of the BMMSCs can be up regulated by the pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid, While the expression of VEGF mRNA,TFPI mRNA,VEGF protein and TFPI protein of the BMMSCs can be down regulated by the pU-VEGF-siRNA recombinant eukaryotic expression plasmid. 3. The conditioned medium after the transfection of the BMMSCs transfected with pcDNA3.1(-)VEGF plasmid can enhance the proliferation of endothelial cell.ObjectiveThis study was firstly to establish an experimental model of atherosclerosis in rats, and then to investigate the changes of the level of VEGF and TFPI protein in the aorta of the atherosclerotic rats which were injected the BMMSCs transfected with pcDNA3.1(-)VEGF and pU-VEGF-siRNA recombinant eukaryotic expression plasmid by caudal vein.Methods1. Sixty six male healthy SD rats were randomly divided into 6 groups. In the control group (A) rats were basic food, while in others group the rats were loaded with high fat diet. The rats were injected with the culture medium (Group A), the culture medium (Group B), BMMSCs (Group C), BMMSCs transfected with pcDNA3.1(-) vector (Group D), BMMSCs transfected with pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid (Group E) and BMMSCs transfected with pU-VEGF-siRNA recombinant eukaryotic expression plasmid (Group F) respectively by caudal vein. 3. The pathological and ultrasonic changes were determined in the aorta of the 58 rats after one week. RT-PCR methods were used to detect the expression of VEGF and TFPI mRNA of the aorta, and Western blot methods were used to detect the levels of VEGF and TFPI protein of the aorta.Results1. No changes were observed in the aorta of control group with ultrasound and pathology, but atherosclerotic plaque was observed in the aorta of experimental group after 3 months. 2. There was no significant difference in the atherosclerotic plaque of the aorta in the rats before and after the BMMSCs treatment. 3. There was no significant difference in the expression of VEGF and TFPI mRNA of aorta in rats received the culture medium treatment, BMMSCs treatment and BMMSCs transfected with pcDNA3.1(-) vector treatment (p＞0.05 vs Control). The expression of VEGF and TFPI mRNA of the aorta in rats received BMMSCs transfected with pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid treatment was significantly (p＜0.05 vs Control). The expression of VEGF and TFPI mRNA of the aorta in rats received BMMSCs transfected with pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid was down regulated (p＜0.05). Treatment with BMMSCs transfected with pU-VEGF-siRNA recombinant eukaryotic expression plasmid significantly increased/decreased the expression of VEGF and TFPI mRNA of the aorta (p＜0.01 vs Control). 4. There were no significant differences in the level of VEGF protein and TFPI protein of aorta of the normal rat rats received saline treatment, BMMSCs treatment and BMMSCs transfected with pcDNA3.1 (-) vector treatment (p＞0.05 vs Control). The expression of VEGF and TFPI mRNA of the aorta in rats received BMMSCs transfected with pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid treatment was significantly (p＜0.05 vs Control). the level of VEGF protein and TFPI protein of the aorta of the atherosclerotic rat model which were injected the BMMSCs transfected with pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid by caudal vein was down regulated and its was significantly less than control groups(p＜0.05). There were significant differences in the level of VEGF protein and TFPI protein of the aorta of groups of the atherosclerotic rat model which were injected the BMMSCs transfected with pcDNA3.1(-) VEGF recombinant eukaryotic expression plasmid by caudal vein and groups of the atherosclerotic rat model which were injected the BMMSCs transfected with pU-VEGF-siRNA recombinant eukaryotic expression plasmid by caudal vein (p＜0.01).ConclusionOur results has provided evidence for the first time that construction of BMMSCs transfected with pcDNA3.1(-)VEGF recombinant eukaryotic expression plasmid is a useful tool to regulate the expression of VEGF and TFPI in the aorta of the atherosclerotic rats.